Compound Having Activity Of Blocking NMDA Receptor Channel, and Pharmaceutical Agent Using The Same

ABSTRACT

To provide a novel compound having NMDA receptor channel blocking activity, and also a pharmaceutical agent comprising the compound. 
     A pharmaceutical agent for the treatment or prevention of a disease caused by overexcitation of an NMDA receptor, which comprises a compound having NMDA receptor channel blocking activity and represented by the formula ( 1 ), a salt thereof, or a hydrate of the compound or the salt.

TECHNICAL FIELD

The present invention relates to compounds having activity of blockingan NMDA receptor channel, and pharmaceutical agents using the same.

BACKGROUND ART

Cerebrovascular disorders such as cerebral infarction and cerebralhemorrhage, and diseases that strongly affect higher brain functionssuch as Alzheimer's disease exhibit poor prognosis, and bring decreasedQOL (Quality of Life) on elderly people and further psychological stresson caretakers. Several pharmaceutical agents for improving brainfunctions have been developed and marketed to date. However, asufficient therapeutic effect has not yet been obtained by using any ofthem. Thus, new and more effective agents for improving brain functionsand agents for preserving brain functions are expected to be developedrapidly.

Meanwhile, N-methyl-D-aspartic acid (hereafter referred to as “NMDA”)receptor, which is one kind of receptor for glutamic acid that is anexcitatory neurotransmitter, is known to transport Ca²⁺ to neuron andthereby strongly participate in memory formation and in development ofsymptoms in cerebral ischemia.

The present inventors already reported in the Non-Patent Document 1shown below that NMDA receptors expressed on Xenopus oocytes followed bycontacting with a plurality of polyamine derivatives and measuring achange in electric potential resulted in a finding that a polyaminederivative having an anthraquinone skeleton showed a significant channelblocking activity (see Non-Patent Document I shown below).

Non-Patent Document 1: J.Pharm. Exp. Ther., 309:884-893

DISCLOSURE OF THE INVENTION

[Problems to be Solved by the Invention]

However, even in the technology described in the above-mentionedNon-Patent Document 1, intensity of channel blocking activity remains tobe improved.

Thus, an object of the present invention is to provide a novel compoundwhich has NMDA receptor channel blocking activity, and a pharmaceuticalagent comprising the compound.

[Means for Solving the Object]

Thus, a means of the present invention relate to, as one means, apharmaceutical agent for use in medical treatment or prevention ofdiseases caused by overexcitation of NMDA receptors, which comprises acompound having NMDA receptor channel blocking activity and representedby the following formula (1), a salt thereof, or a hydrate of thecompound or the salt.

(where, B-ring condensed to A-ring and C-ring is quinone, benzene,pyrrole, pyridine, 1,4-dihydropyridine, azepine, 4,5-dihydroazepine or1,4-thiazine. R₁-R₅ are each independently formyl, acyl, hydrogen,alkyl, alkoxy, halogen, hydroxy, amino, aryl or carboxamido, and atleast either of R₁ or R₂ is a carboxamide group having a polyaminestructure containing three or more amino groups except an amino group ofthe carboxamido group. R₁-R₅ may be identical or different).

Further, in that means, the pharmaceutical agent preferably comprises acompound having NMDA receptor channel blocking activity and representedby the following formula (2), a salt thereof, or a hydrate of thecompound or the salt, but not limited thereto.

Further, in that means, the pharmaceutical agent preferably comprises acompound having NMDA receptor channel blocking activity and representedby the following formula (3), a salt thereof, or a hydrate of thecompound or the salt, but not limited thereto.

Further, in that means, the pharmaceutical agent preferably comprises acompound having NMDA receptor channel blocking activity and representedby the following formula (4), a salt thereof, or a hydrate of thecompound or the salt, but not limited thereto.

Further, in that means, the pharmaceutical agent preferably comprises acompound having NMDA receptor channel blocking activity and representedby the following formula (2), a salt thereof or a hydrate of thecompound or the salt, but not limited thereto.

Disorders caused by overexcitation of NMDA receptors are preferablycerebrovascular disorder, cerebral infarction, cerebral hemorrhage,Alzheimer's disease, central neurodegeneration, traumatic brain damage,traumatic spinal cord injury or demyelinating disease, or sequelae ofthese diseases, but not limited thereto

Further, an another means of the present invention relates to a compoundrepresented by the following formula (1):

(where, B-ring condensed to A-ring and C-ring is quinone, benzene,pyrrole, pyridine, 1,4-dihydropyridine, azepine, 4,5-dihydroazepine or1,4-thiazine. R₁-R₅ are each independently formyl, acyl, hydrogen,alkyl, alkoxy, halogen, hydroxy, amino, aryl or carboxamido, and atleast either of R₁ or R₂ is a carboxamide group having a polyaminestructure containing three or more amino groups except an amino group ofthe carboxamido group. R₁-R₅ may be identical or different).

Further, in that means, the compound is preferably represented by thefollowing formula (2), but not limited thereto.

Further, in that means, the compound is preferably represented by thefollowing formula (3), but not limited thereto.

Further, in that means, the compound is preferably represented by thefollowing formula (4), but not limited thereto.

Further, in that means, the compound is preferably represented by thefollowing formula (5), but not limited thereto.

[Advantage of the Invention]

As mentioned above, the present invention can provide novel compoundshaving NMDA receptor channel blocking activity, pharmaceutical agentscomprising the compounds, and screening methods using the compounds.

DETAILED DESCRIPTION OF THE INVENTION Embodiments

One embodiment of the present invention is a compound having NMDAreceptor channel blocking activity and represented by the followingformula (1):

In the aforementioned formula (1), B-ring condensed to A-ring and C-ringis quinone, benzene, pyrrole, pyridine, 1,4-dihydropyridine, azepine,4,5-dihydroazepine or 1,4-thiazine. R₁-R₅ are each independently formyl,acyl, hydrogen, alkyl, alkoxy, halogen, hydroxy, amino, aryl orcarboxamido, and at least either of R₁ or R₂ is a carboxamide grouphaving a polyamine structure containing three or more amino groupsexcept an amino group of the carboxamido group. R₁-R₅ may be identicalor different.

The present inventors made intensive studies for novel compounds thatshow NMDA receptor channel blocking activity, and found out that apolyamine inhibited NMDA receptor activity and suppressed intracellularcalcium influx. The present inventors made a further detailedinvestigation, and reached a compound represented by the above-mentionedformula (1). The term “polyamine” described herein denotes a compoundhaving two or more amino groups in a molecule. Further, the phrase“polyamine structure” denotes a structure having two or more aminogroups, and includes, but not limited to, putrescine, cadaverine,spermidine, 1,3-diaminopropane, caldine, homospermidine,3-aminopropyl-cadaverine, norspermine, homospermine, thermospermine,caldopentamine. The more preferable polyamine structure is homospermine.“The polyamine structure containing three or more amino groups except anamino group of the carboxamido group” is required for NMDA receptorchannel blocking activity. Homospermine is preferable for such apolyamine structure, but not limited thereto.

The compound represented by the aforementioned formula (1) can besynthesized by a conventional method, as clearly shown in examplesdescribed later, but it is not limited to the synthesized one as far asit is available.

Another embodiment of the present invention is a pharmaceutical agentcomprising as an active ingredient a compound having NMDA receptorchannel blocking activity and represented by the aforementioned formula(1), a salt thereof or a hydrate of the compound or the salt.

The aforementioned pharmaceutical agent can be supplied, in addition tothe aforementioned compound having NMDA receptor channel blockingactivity, with various pharmaceutically acceptable formulationcomponents such as carriers, binders, stabilizers, excipients, diluents,pH buffers, disintegrators, solubilizers, dissolution auxiliaries, andisotonizers. The compound having NMDA receptor channel blocking activitycan be administrated orally or parenterally at an appropriate dosagedepending on gender, body weight and symptoms of a patient forprevention or treatment. That is to say, it can be administrated in aconventional administration form. For example, it can be orallyadministrated in a dosage form such as powders, granules, capsules,syrups and suspensions. Alternatively, it can be parenterallyadministrated in a dosage form for injection such as solutions,emulsions and suspensions, or intranasally administrated in a sprayform.

Consequently, the novel compound having NMDA receptor channel blockingactivity and the pharmaceutical agent comprising the compound can beprovided.

EXAMPLE

The present invention may be explained more particularly with examplesshowing activity of compounds of the aforementioned embodiment; however,the present invention is not limited to these examples.

Production Example 1 Synthesis of Anthraquinone Homospermine (AQ444)

Anhydrous triethylamine (0.46 mL) and para-nitrophenol (238 mg) wereadded to an anhydrous methylene chloride solution (10 mL) ofanthraquinone 2-carbonyl chloride (300 mg), and stirred for 3 hoursunder an argon stream at room temperature. The reaction solution waspoured into an aqueous IN sodium hydroxide solution and extracted withchloroform, 5% methanol/chloroform mixed solution. An organic layer thusobtained was washed with a saturated saline, dried on magnesium sulfate,followed by concentration under a reduced pressure to form solidprecipitate, which was then filtered by suction to yield ayellowish-white solid of anthraquinone 2-nitrophenyl ester (270 mg, 65%yield). ¹H-NMR and ¹³C-NMR spectral data and EI-MS data of theyellowish-white solid are shown below.

¹H-NMR (500 MHz, CDCl₃) δ 9.13 (1H, d, J=1.5 Hz), 8.59 (1H, dd, J=7.9,1.5 Hz), 8.50 (1H, d, J=7.9 Hz), 8.40-8.36 (4H, overlapped), 7.88(2H,m), 7.50 (2H,m).

¹³C-NMR (125 MHz, CDCl₃) δ 182.3, 182.1, 162.8, 155.2, 145.7, 136.9,135.1, 134.72, 134.66, 133.8, 133.5, 133.3, 129.3, 128.0, 127.60,127.56, 125.4, 122.5.

EI-MS m/z (%) 373(M⁺, 1), 235 (100).

Next, an anhydrous chloroform solution (2.5 mL) of anthraquinone2-paranitrophenyl ester (11 mg) was added to an anhydrous chloroformsolution (2.5 mL) of 5,10-bis(2-nitrobenzene sulfonyl)-5,10-diaza-1,14-tetradecanediamine (36 mg), and stirred for 4 hours under an argonstream at room temperature, and then for 1 hour at 50° C. The reactionsolution was directly applied to an amino silica gel column(methanol/chloroform mixed solution) for purification to get a 2%methanol/chloroform eluate, which was then concentrated under a reducedpressure to yield an AQ444 derivative (15 mg, 60% yield). The tetraaminederivative used in large excess as a raw material was recovered. ¹H-MNRspectral data, ¹³C-NMR spectral data, and FAB-MS data of the AQ44derivative are shown below.

¹H-NMR (400 MHz, CDCl₃) δ 8.59 (1H, d, J=1.7 Hz), 8.33 (1H, d, J=8.1Hz), 8.31 (2H, m), 8.25 (1H, d, J=8.1, 1.7 Hz), 7.99 (2H, m), 7.83 (2H,m), 7.72-7.60 (6H, overlapped), 3.51 (2H, m), 3.33-3.22 (8H,overlapped), 2.64 (2H, dd, J=7.0, 7.0 Hz), 1.72-1.44 (10H, overlapped),1.35 (2H, m).

¹³C-NMR (150 MHz, CDCl₃) δ182.6, 182.0, 165.9, 148.0, 139.8, 135.1,134.5, 134.4, 133.62, 133.56, 133.5, 133.4, 133.3, 133.1, 131.8, 131.7,130.72, 130.68, 127.9, 127.5, 125.3, 124.2, 47.6, 47.5, 47.4, 46.9,45.4, 41.7,39.7, 30.6, 26.6, 26.0, 25.6, 25.4, 25.1.

FAB-MS (NBA) m/z 835 (MH⁺).

Then, calcium carbonate (24 mg) and thiophenol (7 μL) were added to ananhydrous DMF solution (1.0 mL) of the AQ444 derivative (48 mg) obtainedas mentioned above, and stirred for 13 hours under an argon stream atroom temperature. The reaction solution was directly applied to an aminosilica gel column (methanol/chloroform mixed solution) for purificationto get 5%, 10% and 20% methanol/chloroform eluates, which were thenconcentrated under a reduced pressure to yield AQ444 (19 mg, 70% yield).The Structural formula (2) of AQ444 thus obtained is shown below as wellas UV spectrum, ¹H-NMR spectral data, ¹³C-NMR spectral data, and FAB-MSdata.

UV (MeOH) λ_(max)326.0, 256.5, 210.0 nm.

¹H-NMR (500 MHz, CDCl₃) δ8.60 (1H, br-d, J=1.5 Hz), 8.38 (1H, d, J=8.1Hz), 8.37-8.30 (4H, overlapped), 7.83 (2H, m), 3.52 (1H, br-ddd, J=5.9,5.9, 5.9 Hz), 2.72 (2H, dd, J=6.5, 6.5 Hz), 2.67 (1H, dd, J=6.6, 6.6Hz), 2.65-2.60 (3H, overlapped), 2.56-2.50 (4H, overlapped), 1.78 (2H,br-quin, J=6.3 Hz), 1.67 (2H, br-quin, J=6.3 Hz), 1.64-1.40 (8H,overlapped).

¹³C-NMR (125 MHz, CDCl₃) δ182.6, 182.5, 165.7, 140.2, 134.9, 134.42,134.35, 133.4133.3, 127.8, 127.4, 127.3, 125.1, 51.9, 49.8, 49.7, 49.6,49.1, 42.1, 40.3,36.3, 31.5, 27.9, 27.8, 27.7, 27.4.

FAB-MS (NBA+NaCl) m/z 465 (MH).

Production Example 2 Synthesis of Carbazole Spermine

Anhydrous triethylamine (546 μL) and para-nitrophenol (272 mg) wereadded to an anhydrous methylene chloride solution (10 mL) of carbazoleN-carbonyl chloride (300 mg), and stirred for 3.5 hours under an argonstream at room temperature. The reaction solution was poured into anaqueous 1N sodium hydroxide solution and extracted with chloroform, 5%methanol/chloroform mixed solution. An organic layer thus obtained waswashed with a saturated saline, dried on magnesium sulfate, and thenconcentrated under a reduced pressure to a yield yellowish-white solidof carbazole N-paranitrophenyl ester (401 mg, 92% yield). ¹H-MNRspectral data of the yellowish-white solid thus obtained is shown below.

¹H-NMR (400 MHz, CDCl₃) δ8.39 (2H, d-like, J=10.1 Hz), 8.32 (2H, d-like,J=7.5 Hz), 8.03 (2H, br-d, J=7.5 Hz), 7.57 (2H, d-like, J=10.1 Hz), 7.52(2H, ddd, J=7.5, 7.5, 1.2 Hz), 7.45 (2H, ddd, J=7.5, 7.5, 1.2 Hz).

Next, an anhydrous chloroform solution (6.5 mL) of carbazoleN-paranitrophenyl ester (30 mg) was added to an anhydrous chloroformsolution (6.5 mL) of spermine (40 mg), and stirred for 3.5 hours underan argon stream at room temperature. The reaction solution was directlyapplied to an amino silica gel column (methanol/chloroform mixedsolution) for purification to get a 5% methanol/ chloroform eluate,which was then concentrated under a reduced pressure to yield carbazolespermine (25 mg, 70% yield). The structural formula (3) of carbazolespermine thus obtained is shown below, as well as ¹H-NMR spectral data,¹³C-NMR spectral data, and FAB-MS data.

¹H-NMR (500 MHz, CDCl₃) δ8.08 (2H, d, J=7.8 Hz), 8.02 (2H, d, J=7.8 Hz),7.45 (2H, ddd, J=7.8, 7.8, 0.9 Hz), 7.31 (2H, ddd, J=7.8, 7.8, 0.9 Hz),3.69 (2H, br-dd, J=10.4, 5.8 Hz), 2.83 (2H, br-dd, J=5.6, 5.6 Hz), 2.69(2H, dd, J=6.9, 6.9 Hz), 2.52 (4H, overlapped), 2.35 (2H, br-dd, J=6.7,6.7 Hz), 1.84 (2H, br-quin, J=6.0 Hz), 1.54 (2H, quin, J=6.7 Hz),1.28-1.19 (4H, m).

¹³C-NMR (125 MHz , CDCl₃) δ152.8, 138.3, 126.6, 124.8, 121.8, 119.9,113.8, 49.8, 49.7, 49.3, 47.7, 41.6, 40.5, 33.6, 28.1, 27.6, 27.5.

FAB-MS (NBA) m/z 396 (MH⁺).

Production Example 3 Synthesis of Dibenzoazepine Homospermine (DBA444)

An anhydrous methylene chloride solution (1.5 mL) of commerciallyavailable 5H-dibenzo [b,f] azepine-5-carbonyl chloride (24.5 mg) wasadded to an anhydrous methylene chloride solution (6.5 mL) of 5,10-bis(2-nitrobenzene sulfonyl)-5, 10-diaza-1,14-tetradecane diamine (115 mg),and stirred for 2 hours under an argon stream at room temperature. Thereaction solution was directly applied to amino silica gel column(methanol/chloroform mixed solution) for purification to get an 1%methanol/chloroform eluate, which was then concentrated under a reducedpressure to yield a DBA444 derivative (60.1 mg, 76% yield). Further, thetetraamine derivative used in large excess as a raw material wasrecovered. ¹H-NMR spectral data, ¹³C-NMR spectral data, and FAB-MS dataof the DBA444 derivative are shown below.

¹H-NMR (400 MHz, CDCl₃) δ7.98-7.94 (2H, m), 7.70-7.63 (4H, overlapped),7.61-7.57 (2H, m), 7.45-7.40 (4H, overlapped), 7.40-7.30 (4H,overlapped), 6.92 (2H, s), 4.35 (1H, br-dd, J=5.8, 5.8 Hz), 3.26-3.22(6H, overlapped), 3.19 (2H, dd, J=7.6, 7.6 Hz), 3.09 (2H, br-ddd, J=6.5,6.5, 6.5 Hz), 2.65 (2H, dd, J=7.0, 7.0 Hz), 1.58-1.31 (12H, overlapped).

¹³C-NMR (100 MHz, CDCl₃) δ156.3, 147.96, 147.94, 140.0, 135.2, 133.4,133.3, 131.70, 131.65, 130.60, 130.55, 130.4, 129.6, 129.4, 129.1,127.6, 124.1, 124.0, 47.3, 47.0, 46.8, 46.7, 41.6, 39.7, 30.5, 27.3,25.6, 25.3, 25.0.

FAB-MS (NBA) m/z 820 (MH⁺).

Then, calcium carbonate (92.9 mg) and thiophenol (55.2 μL) were added toan anhydrous DMF solution (4.5 mL) of the DBA444 derivative (183.5 mg)obtained as mentioned above, and stirred for 2.5 hours under an argonstream at room temperature. The reaction solution was directly appliedto amino silica gel column (methanol/chloroform mixed solution) forpurification to get a 5% methanol/chloroform eluate, which was thenconcentrated under a reduced pressure to yield DBA444 (62.4 mg, 62%yield). The structural formula (4) of DBA444 thus obtained is shownbelow as well as data of UV spectrum, ¹H-NMR spectral data, ¹³C-NMRspectral data, and FAB-MS data.

UV (MeOH) λ_(max)285.5, 247.5 (sh), 213.5 nm.

¹H-NMR (400 MHz, CDCl₃) δ7.46-7.40 (4H, overlapped), 7.38-7.30 (4H,overlapped), 6.91 (2H, s), 4.46 (1H, br-dd, J=5.7, 5.7 Hz), 3.13 (2H,br-ddd, J=6.3, 6.3, 6.3 Hz), 2.70 (2H, dd, J=6.7, 6.7 Hz), 2.63-2.59(2H, overlapped), 2.57-2.53 (2H, overlapped), 1.56-1.46 (8H,overlapped), 1.43-1.37 (4H, overlapped).

¹³C-NMR (100 MHz, CDCl₃) δ156.3, 140.1, 135.3, 130.4, 129.6, 129.3,129.2, 127.5, 49.74, 49.72, 49.6, 49.3, 42.0, 40.4, 31.5, 27.94, 27.85,27.84, 27.4, 27.1.

FAB-MS (NBA) m/z 450 (MH⁺).

Production Example 4 Synthesis of Dihydrodibenzoazepine Homospermine(DDBA444)

An anhydrous methylene chloride solution (8.0 mL) of commerciallyavailable 10,11-dihydro-dibenzo [b,f] azepine-5-carbonyl chloride (84.9mg) was added to an anhydrous methylene chloride solution (22.0 mL) of5,10-bis (2-nitrobenzene sulfonyl)-5,10-diaza-1,14-tetradecanediamine(396.0 mg), and stirred for 19 hours under an argon stream at roomtemperature. The reaction solution was directly applied to an aminosilica gel column (methanol/chloroform mixed solution) for purificationto get a 1% methanol/chloroform eluate, which was then concentratedunder a reduced pressure to yield a DDBA444 derivative (257.5 mg, 95%yield). Further, the tetraamine derivative used in large excess as a rawmaterial was recovered. ¹H-NMR spectral data, ¹³C-NMR spectral data, andFAB-MS data of the DDBA444 derivative are shown below.

¹H-NMR (400 MHz, CDCl₃) δ7.98-7.95 (2H, m), 7.70-7.64 (4H, overlapped),7.61-7.56 (2H, m), 7.36-7.33 (2H, m), 7.22-7.20 (6H, overlapped), 4.60(1H, br-dd, J=6.0, 6.0 Hz), 3.26-3.15 (10H, overlapped), 2.65 (2H, dd,J-6.8, 6.8 Hz), 1.58-1.33 (16H, overlapped).

¹³C-NMR (100 MHz, CDCl₃) δ156.4, 147.9, 140.5, 137.2, 133.4, 133.3,131.69, 131.64, 130.6, 130.5, 130.3, 128.9, 127.8, 127.0, 124.1, 124.0,47.3, 47.0, 46.8, 46.7, 41.5, 39.9, 30.7, 30.5, 27.4, 25.5, 25.4, 25.0.

FAB-MS (NBA) m/z 822 (MH⁺).

Then, cesium carbonate (237.8 mg) and thiophenol (59.9 μL) were added toan anhydrous DMF solution (4.5 mL) of the DDBA444 derivative (200.0 mg)obtained as mentioned above, and stirred for 3 hours under an argonstream at room temperature. The reaction solution was directly appliedto an amino silica gel column (methanol/chloroform mixed solution) forpurification to get a 5% methanol/chloroform eluate, which was thenconcentrated under a reduced pressure. The residues thus obtained wereapplied to an alumina column (ethanol/chloroform/concentrated ammoniawater [=5/95/1] mixed solution) for purification to get an eluate, whichwas then concentrated under a reduced pressure to yield DDBA444 (68.8mg, 63% yield). The structural formula (5) of DDBA444 thus obtained isshown below as well as UV spectrum, ¹H-NMR spectral data, ¹³C-NMRspectral data, and FAB-MS data.

UV (MeOH) λ_(max)239.5 (sh), 203.0 nm.

¹H-NMR (400 MHz, CDCl₃) δ7.38-7.36 (2H, m), 7.23-7.20 (6H, overlapped),4.69 (1H, br-dd, J-5.4, 5.4 Hz), 3.22 (2H, br-ddd, J=6.3, 6.3, 6.3 Hz),2.70 (2H, dd, J=6.5, 6.5 Hz), 2.63-2.54 (8H, overlapped), 1.63-1.56 (4H,overlapped), 1.53-1.43 (12H, overlapped).

¹³C-NMR (150 MHz, CDCl₃) δ156.5, 140.7, 137.3, 130.4, 129.1, 127.8,127.1, 49.90, 49.87, 42.2, 40.7, 31.6, 30.9, 28.2, 28.0, 27.5, 27.3.

FAB-MS (NBA) m/z 452 (MH⁺).

Example 1 Investigation of Channel Blocking Activity of AQ444 on NMDAReceptors Expressed on Xenopus oocytes, and of its Voltage Dependence

The cDNA clones for NMDA receptors were used to prepare the cRNAs, whichwere then injected in Xenopus oocytes. The cRNAs of subtype 1 (NR1) andsubtype 2 (NR2A) of NMDA receptors were injected in a Xenopus oocyte ina ratio of 1:5 (0.1 ng of NR1 and 0.5 ng of NR2A) and incubated in anincubator for several days to express the NMDA receptors on the membraneof the developed egg. The expression was confirmed by a two electrodevoltage clamp system. A glass capillary was filled with a 3M potassiumchloride solution for use as an electrode, which was then inserted intoan egg placed in a perfusion solution to measure a change in membranepotential. The perfusion solution used comprised 100 mM sodium chloride,2 mM potassium chloride, 1.8 mM barium chloride and 10 mM HEPES, pH 7.5.The electric current through the NMDA receptor in the presence of 10 μMglycine and 10 μM glutamic acid was used as a standard (control) toinvestigate the channel blocking activity of AQ444. Results are shown inFIG. 1. FIG. 1 (A) shows the results determined under membrane potentialclamp at −70 mV. In the figure, the bars denote the timing periodsduring which glycine and glutamic acid (Gly +Glu) were present with orwithout the AQ444 added, respectively. In FIG. 1 (A), time and electriccurrent value are denoted along the horizontal and vertical axes,respectively.

As a result as shown in FIG. 1 (A), AQ444 showed channel blockingactivity on the NR1/NR2A with the 50% inhibition concentration (IC₅₀) of0.47 μM. This value revealed that AQ444 has about two times strongerchannel blocking activity than memantine (IC₅₀=0.95 μM) that iscommercially available as a cerebral function improving agent.

As shown in FIG. 1 (B), it was found that the channel blocking activityof AQ444 showed voltage dependence according to continuous change inmembrane potential of the egg from −150 mV to +10 mV. Since the membranepotential of a cell is normally about −70 mV, it was revealed that AQ444worked on the cells with static membrane potential and showed channelblocking activity on NMDA receptor.

Example 2 Investigation of Reactivity of AQ444 with each the NMDAReceptor Subtypes

In the same method as shown in Example 1, eggs were injected with thecRNAs for NR1/NR2A (0.1 ng of NR1 and 0.5 ng of NR2A), those forNR1/NR2B (0.1 ng of NR1 and 0.5 ng of NR2B), those for NR1/NR2C (4 ng ofNR1 and 20 ng of NR2C), or those for NR1/NR2D (4 ng of NR1 and 20 ng ofNR2D) to investigate the reactivity of AQ444 with each the receptorsprepared. AQ444 was added to the perfusion solution to get a finalconcentration of 0.1 to 30 μM. Results obtained are shown in FIG. 2.AQ444 showed channel blocking activity more selectively againstNR1/NR2A, NR1/NR2B and NR1/NR2D. Further, the intensities in channelblocking activity against the receptors were in the order ofNR1/NR2A=NR1/NR2B=NR1/NR2D>>NR1/NR2C.

Example 3 Investigation of AQ444 Channel Blocking Activity on NMDAReceptor Mutants with Amino Acids Substitutions

Site-directed mutagenesis was utilized to construct NMDA receptormutants with amino acid substitutions. The NMDA receptor mutants wereexpressed in eggs in the same method as shown in Example 1 toinvestigate for channel blocking activity of AQ444. Results obtained areshown in FIG. 3. As shown in FIG. 3, both an NR1 mutant withsubstitution of asparagine for glutamine at position 616 (hereinafter,abbreviated to N616Q) and an NR1 mutant N616R showed significantlydecreased channel blocking activity. NR2B mutants, that are N615Q andN616Q, also showed significantly decreased channel blocking activity.These residues are located at the site in the M2 region of the NMDAreceptor which is considered to define the size of a channel pore,suggesting that they are strongly associated with the channel blockingactivity of AQ444. Other relevant amino acid residues on the NMDAreceptors are shown in FIG. 4 together with their models.

Example 4 Channel Blocking Activity of Carbazole Spermine on NMDAReceptors Expressed on Xenopus Oocytes

In the same method as shown in Example 1, carbazole spermine was used inreplace of AQ444 to determine its channel blocking activity. As aresult, the IC₅₀ of carbazole spermine was 15 μM. This value revealedthat carbazole spermine has about six times stronger channel blockingactivity than memantine (IC₅₀=0.95 μM) which is commercially availableas a cerebral function improving agent.

DBA444 and DDBA444 containing an azepine ring and a 4,5-dihydroazepinering respectively inhibited the NMDA receptor activity in avoltage-dependent manner. The both compounds had their respective IC₅₀sof 0.24 and 0.072 μM at −70 mV. Consequently, it was found that the bothcompounds showed about 4 times and 13 times stronger channel blockingactivity respectively than memantine. Further, DBA444 and DDBA444 hadtheir respective IC₅₀s of 132 μM and 133 μM in cytotoxicity, which werecomparable to memantine with IC₅₀ of 147 μM. Therefore, DBA444 andDDBA444 were expected to effect with a smaller amounts and less sideeffect than memantine.

As demonstrated above, it was revealed that the present invention canprovide a novel compound having NMDA receptor channel blocking activityand a pharmaceutical agent comprising the compound.

INDUSTRIAL APPLICABILITY

Along with the aging society, the population of people suffering fromdiseases which bring impairment in higher cerebral functions, such ascerebrovascular disorders and Alzheimer's disease, has been increasingyear by year. A compound provided by the present invention and apharmaceutical agent using the same have industrial applicability to beused for an agent of preserving brain functions. There is no doubt thatthe novel agent of preserving brain functions thus developed would bringimproved QOL (Quality of Life) to the patients, and moreover, to thecaretakers, and thus would make an immensurable contribution in medicaltreatment for the elderly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) shows a waveform indicating that addition of AQ444 resultedin suppression of current flow through the NMDA receptors expressed onXenopus oocytes.

FIG. 1 (B) is a graph showing voltage-dependent channel blockingactivity of AQ444.

FIG. 2 is a graph showing channel blocking activity of AQ444 on each theNMDA receptor subtypes.

FIG. 3 is a graph showing channel blocking activity of AQ444 on variousNMDA receptor mutants.

FIG. 4 shows the predicted structural models of NMDA receptors aroundtheir channel pores with indicating the amino acid residues involved inchannel blocking activity of AQ444.

1. A pharmaceutical agent for use in medical treatment or prevention ofdiseases caused by the overexcitation of an NMDA receptor, comprising acompound having NMDA receptor channel blocking activity and representedby the following formula (1), a salt thereof, or a hydrate of thecompound or the salt.

where, B-ring condensed to A-ring and C-ring is quinone, benzene,pyrrole, pyridine, 1,4-dihydropyridine, azepine, 4,5-dihydroazepine or1,4-thiazine, R₁-R₅ are each independently formyl, acyl, hydrogen,alkyl, alkoxy, halogen, hydroxy, amino, aryl or carboxamido, and atleast either of R₁ or R₂ is a carboxamide group having a polyaminestructure containing three or more amino groups except an amino group ofthe carboxamido group, and R₁-R₅ are identical or different.
 2. Thepharmaceutical agent according to claim 1, comprising a compound havingNMDA receptor channel blocking activity and represented by the followingformula (2), a salt thereof, or a hydrate of the compound or the salt.


3. The pharmaceutical agent according to claim 1, comprising a compoundhaving NMDA receptor channel blocking activity and represented by thefollowing formula (3), a salt thereof, or a hydrate of the compound orthe salt.


4. The pharmaceutical agent according to claim 1, comprising a compoundhaving NMDA receptor channel blocking activity and represented by thefollowing formula (4), a salt thereof, or a hydrate of the compound orthe salt.


5. The pharmaceutical agent according to claim 1, comprising a compoundhaving NMDA receptor channel blocking activity and represented by thefollowing formula (5), a salt thereof, or a hydrate of the compound orthe salt.


6. The pharmaceutical agent according to claim 1, wherein said diseasescaused by the overexcitation of the NMDA receptor are cerebrovasculardisorder, brain infarction, cerebral hemorrhage, Alzheimer's disease,central neurodegeneration, traumatic brain damage, traumatic spinal cordinjury or traumatic demyelinating disease, or sequelae of thesediseases.
 7. A compound represented by the following formula (1).

where, B-ring condensed to A-ring and C-ring is quinone, benzene,pyrrole, pyridine, 1,4-dihydropyridine, azepine, 4,5-dihydroazepine or1,4-thiazine, R₁-R₅ are each independently formyl, acyl, hydrogen,alkyl, alkoxy, halogen, hydroxy, amino, aryl or carboxamido, and atleast either of R₁ or R₂ is a carboxamide group having a polyaminestructure containing three or more amino groups except an amino group ofthe carboxamido group, and R₁-R₅ maybe are identical or different. 8.The compound according to claim 7, which is represented by the followingformula (2).


9. The compound according to claim 7, which is represented by thefollowing formula (3).


10. The compound according to claim 7, which is represented by thefollowing formula (4).


11. The compound according to claim 7, which is represented by thefollowing formula (5).